A review of factors influencing maturation of Atlantic salmon (Salmo salar) with focus on water recirculation aquaculture system environments

Transcription

1 A review of factors influencing maturation of Atlantic salmon (Salmo salar) with focus on water recirculation aquaculture system environments October 2015 Submitted to: Salmon Aquaculture Innovation Fund Tides Canada W Hastings St. Vancouver, BC V6B 1H5 catherine.emrick@tidescanada.org Submitted by: Christopher Good, Director of Aquatic Veterinary Research John Davidson, Senior Research Associate The Conservation Fund Freshwater Institute 1098 Turner Rd. Shepherdstown, WV - USA This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives License: The Conservation Fund, some rights reserved.

2 EXECUTIVE SUMMARY Maturation of Atlantic salmon Salmo salar is an extremely complex process, particularly in aquaculture systems, with many variables (known or otherwise) having the capacity to influence the timing and prevalence of maturation, and acting as promoters and/or inhibitors of sexual development. The vast majority of research carried out on salmon maturation in aquaculture has been in context of traditional culture; namely, land-based smolt production in freshwater systems, followed by transfer to sea cages for growout. However, very little research has specifically examined salmon maturation in land-based, closed containment water recirculation aquaculture systems (RAS), which have recently received attention as an alternative technology for the sustainable production of market-size Atlantic salmon. Given the high prevalence of grilsing observed in the nascent closed containment salmon industry, and the potential economic challenges facing its expansion, it is imperative that best management practices are developed to reduce economic losses and other deleterious outcomes resulting from maturation. This review provides a brief summary of published research on factors associated with early salmon maturation, as well as information on current practices applied in closed containment operations to minimize grilsing. In light of the novelty of raising salmon to market size in a closed containment environment, and the paucity of research on maturation in such environments, it is difficult at present to make specific recommendations to reduce grilsing in RAS; however, much-needed research can be directed, to an extent, based on findings made in traditional settings, as well as early observations made at closed containment operations producing market-size Atlantic salmon. Among the numerous environmental variables that reportedly influence salmon maturation, photoperiod and water temperature are foremost; while photoperiod generally influences the decision to undergo or delay Page 2

3 maturation (made months in advance), water temperature tends to control the magnitude of maturation in a given salmon population. Hence, the warmer water temperatures typically maintained in existing RAS growout operations could be contributing to increased grilsing; however, baseline research on water temperature, in conjunction with other variables such as photoperiod, salinity, and feed energy content / fish adiposity, needs to be carried out in order to make definitive recommendations that will guide best management practices for closed containment salmon operations. Research is also needed on other potential influencers of maturation that are specific to RAS, such as the degree of exercise provided in circular tanks, the accumulation and impact of waterborne steroid hormones, and other water quality elements including endocrine disrupting compounds. It must be noted that key variables that may promote maturation are also associated with improved growth performance, and therefore recommendations to reduce maturation could be at odds with current production practices. Given the complexity of the overall problem, and the potential conflict between reducing early maturation, while optimizing Atlantic salmon growth performance, the most expedient solution at present is for industry to partner with breeders to promote the availability of an all-female germplasm for closed containment operators. There is already a possibility that all-female eggs will be available from a commercial supplier in Iceland beginning in late If this becomes a reality (and all-female eggs are available to producers on a regular basis) it would avoid the significant research necessary to develop effective grilse reduction protocols for mixed sex populations. However, female grilsing, while not currently a major problem but still present to a degree in specific operations would also need to be investigated, and remedial best management practices developed. Page 3

4 TABLE OF CONTENTS Introduction...5 Life history of Atlantic salmon...7 Factors influencing maturation...13 Photoperiod...13 Water temperature...25 Feed...35 Exercise...37 Genetic strain...39 Water chemistry Nitrate nitrogen...42 Other endocrine disrupting compounds Steroid hormones...46 Salinity Summary...51 References...53 Tables & Figures...69 Page 4

5 INTRODUCTION The development of sexual maturation in Atlantic salmon Salmo salar is a complex, multifactorial process. The extreme variability of age and size at maturation observed for this species is considered the result of evolutionary adaptation within various river and ocean environments to maximize reproductive success. Although beneficial to the species in its natural environment, this variability in maturation timing can pose a significant problem to aquaculturists. Specifically, early maturing Atlantic salmon (i.e. grilse ) often exhibit decreased growth and feed conversion efficiency (McClure et al., 2007), reduced product quality (Aksnes et al., 1986), increased susceptibility to opportunistic microorganisms (St-Hilaire et al., 1998), and, overall, represent a major source of economic loss for farmers (Johnston et al., 2006; McClure et al., 2007). In the Canadian Maritime salmon farming industry (estimated gross revenue of $250 million CAD in 2002), grilsing was estimated to represent $11-24 million in lost revenue (McClure et al., 2007), with in-cage prevalence of grilsing estimated at 20-30% between (Peterson et al., 2003). Over the years, the traditional salmon farming industry has adopted various strategies to reduce grilsing; these include photoperiod control (Bromage et al., 2001), selective breeding for late maturation (Gjedrem, 2000), and induced triploidy during egg incubation (Benfey, 1999). Overall, these efforts have been successful at reducing early salmon maturation; however, grilsing still remains a significant problem in certain regions of the world. With the development and implementation of next-generation technologies to produce salmon, i.e. land-based, closed containment operations utilizing water recirculation aquaculture systems (RAS), the issue of precocious maturation has returned to the forefront among factors affecting production and profitability of these new operations. During Atlantic salmon Page 5

6 growout trials conducted at The Conservation Fund s Freshwater Institute (TCFFI), precocious male maturation has been as high as 80%, with most grilse developing before the mean population weight reaches 2 kg. Anecdotally, early maturation has also been prevalent at other land-based, closed containment facilities raising Atlantic salmon to market size. Given the considerable up-front capital investment required to build and commission a closed containment growout facility, issues that affect profitability, such as early maturation, need to be investigated to improve the likelihood of economic success for these operations. Among the numerous benefits of closed containment technologies is the relatively high degree of control over the culture environment (Summerfelt and Vinci, 2008); therefore, it should be possible to control, refine, or eliminate environmental triggers of early maturation, once they have been identified. Another benefit of closed containment operations, however, is improved fish growth performance through environmental optimization (e.g. relatively high and constant water temperature, near-saturation dissolved oxygen levels, and 24-hour automated feeding regimes) which, as will be discussed, could also instigate early maturation. At this early stage in the development of a land-based, closed containment Atlantic salmon industry, it is imperative to address and solve issues such as early maturation in order to facilitate industry expansion and to meet the growing global demand for sustainable salmon. The purpose of this paper is to review previous and ongoing scientific research investigating early sexual maturation in Atlantic salmon, with a focus on RAS technologies and the unique production environments that they provide. Current environmental manipulation protocols used in industry to reduce precocious maturation will be discussed, as will the status of other potential Page 6

7 solutions (e.g. all-female germplasm). Finally, based on these reviews, recommendations for specific areas of research will be provided. LIFE HISTORY OF ATLANTIC SALMON To better understand the phenomenon of early maturation in Atlantic salmon, as it relates to intrinsic and extrinsic variables, some background on the reproductive and evolutionary biology of the species is required. This information will provide insight into the natural behaviors and strategies for maturation that are ingrained in Atlantic salmon, and will serve as a basis for understanding the mechanisms controlling maturation in captive populations cultured in land-based, recirculation aquaculture systems. Understanding the remarkably complex life cycle of Atlantic salmon begins with examination of the life history of this species. In context of this review, life history is defined as the sequence and timing of key life events, particularly those related to developmental biology, reproductive behavior, survivorship of offspring, and perpetuance of species, as influenced by the process of natural selection. Atlantic salmon exhibit a highly plastic and diverse range of life history forms that is unmatched by most vertebrates (Hutchings and Jones, 1998). Variation in life history traits, such as time of freshwater habitation and size/age at smoltification (Randall et al., 1987; Metcalfe and Thorpe, 1990; Økland et al., 1993), time of ocean residency and age at reproductive maturity (Scarnecchia, 1983; Saunders, 1986; Thorpe, 1986), and adult size at maturity (Hutchings and Morris, 1985; Saunders, 1986), among other variables (Youngson et al., 1983; Klementsen et al., 2003; Reid and Chaput, 2012), has been widely documented. Despite this diversity in life history tactics, most Atlantic salmon are anadromous and generally conform to a relatively similar Page 7

8 pattern of key life events. Therefore, a basic explanation of the typical Atlantic salmon life cycle is provided as the foundation for exploring the divergence of life history traits within this general strategy. Adult Atlantic salmon typically spawn during the fall and early winter within freshwater tributaries of the Atlantic Ocean, depositing and burying eggs in a gravel nest or redd. After a relatively long incubation period lasting into the spring the eggs hatch, and the emergent larvae or alevins rely on endogenous nutrition from a yolk sac for several months. When the alevins have exhausted their yolk reserves, the young fish (now known as fry) leave the redd to begin feeding. The juvenile salmon then develop into parr with laterally oriented vertical bars or stripes (parr marks) that provide camouflage. From the time of hatching, juvenile Atlantic salmon remain in the freshwater habitat for a period lasting one year or longer (Metcalfe and Thorpe, 1990; Økland et al., 1993). Prior to migrating to the ocean, salmon parr undergo a series of morphological and physiological changes that enable adaptation from freshwater to seawater, a process commonly known as smoltification (Hoar, 1976; Folmar and Dickoff, 1980; Wedemeyer et al., 1980; Stefansson et al., 2008). During this metamorphosis, parr marks fade, fin margins darken, and the body becomes more streamlined with a bright, silvery appearance (Folmar and Dickoff, 1980; Wedemeyer et al., 1980). Physiologically, Atlantic salmon smolts develop the ability for hypoosmotic regulation and associated ion regulation that in turn facilitates seawater adaptation (Folmar and Dickoff, 1980; Wedemyer, 1980; Stefansson et al., 2008). Smolts journey from their native rivers, usually in the spring, to specific locations in the Atlantic Ocean where they begin to feed on the rich marine food supply and grow rapidly as they advance towards reproductive maturity. When Atlantic salmon become sexually mature, the Page 8

9 parental migration pattern is repeated, with adults returning to the native streams and rivers from which they hatched to commence spawning. Unlike most Pacific salmonids, adult Atlantic salmon are iteroparous, meaning that they are capable of surviving the rigors of returning to the ocean and recommencing the spawning migration in subsequent year(s) (Ducharme, 1969). While this general pattern of key life events is similar for most Atlantic salmon, many variations in life history traits exist within this strategy, both among and within populations, such as: the duration of freshwater occupancy and age at smoltification (Randall et al., 1987; Økland et al., 1993), the time of ocean residency and age at reproductive maturity (Scarnecchia, 1983; Saunders, 1986; Thorpe, 1986), and adult size at maturity (Hutchings and Jones, 1985; Saunders, 1986). Other documented variations in life history traits for Atlantic salmon include fecundity and egg size (Reid and Chaput, 2012), migratory behavior (Youngson et al., 1983), and non-anadromous versus anadromous forms (Berg, 1985). Marschall et al. (1998) and Klementsen et al. (2003) provide detailed reviews of the aforementioned life history characteristics, among others. For purposes of this report, several life history traits that distinctly relate to early maturation, including age/size at smoltification, as well as sea-age and size at reproductive maturity, will be summarized. Discussion of smoltification is relevant in the context of maturation, because this process represents a transition to the growth period of the life cycle and subsequent progression towards adulthood. For example, for most Atlantic salmon migrating to sea, important decisions are made regarding reproductive development during the months following smoltification (Mangel and Satterwaite, 2008). Page 9

10 Juvenile Atlantic salmon are known to remain in freshwater for a wide period of time ranging from 1-8 years (Metcalfe and Thorpe, 1990). Økland et al. (1993) found that smolt age varied from 2 to 6 years for Atlantic salmon populations in four Norwegian rivers. The duration of freshwater residency and commencement of the parr-smolt transformation has been correlated with achievement of a specific size and/or level of fitness that corresponds to increased marine survival (Stefansson et al., 2008). Numerous studies have described a bimodal length or size distribution that corresponds with smoltification, where faster growing Atlantic salmon parr become smolts after one year, while slower growing parr require an additional year or more to smolt (Thorpe, 1977; Thorpe et al., 1982; Kristinsson et al., 1985; Rowe and Thorpe, 1991; Økland et al., 1993). Thorpe (1977) determined that there was a strong genetic influence on the bimodal distribution of smoltification; however, other factors, particularly environmental cues such as photoperiod (McCormick et al., 1987, Björnsson et al., 1989; Solbakken et al., 1994), are also important. Eriksson and Lundqvist (1982) found evidence of an innate timing system for smoltification in Atlantic salmon and concluded that photoperiod acts to synchronize the parr-smolt transformation. Of the anadromous populations, wide variation also exists relative to the duration of adult residency at sea and age at first maturity, with some salmon overwintering for just one year (typically referred to as grilse) and other groups spending 3-5 winters in the ocean before returning to their natal rivers to spawn (Saunders, 1986; Hutchings and Jones, 1998). Grilse commonly weigh 1-3 kg, while adult salmon spending multiple winters at sea can range from 3-12 kg (Saunders, 1986). Male parr maturation, which typically coincides with continued freshwater residency, is also utilized as a viable Page 10

11 reproductive strategy by a percentage of individuals within many Atlantic salmon populations (Saunders et al., 1982; Myers et al., 1986; Stefansson et al., 2008). Some male Atlantic salmon parr have been found to mature when they are only 10 cm in length (Fleming, 1996). It is important to note that both precocious parr and grilse maturation have been found to be at least partially heritable, as well as controlled by the environment (Naevdal, 1983; Myers et al., 1986; Herbinger and Friars, 1991; Marshcall et al., 1998). The wide variation of reproductive tactics, particularly age at maturity, could be an evolutionary strategy designed to maintain biodiversity and genetic contribution of a cohort over a number of years (Saunders and Schom, 1985). Overall, the diverse range and plastic nature of life histories displayed by Atlantic salmon is likely an evolutionary adaptation to optimize reproductive success and to perpetuate the species (Fleming, 1996; Thorpe et al., 1998). In short, the life cycle of the Atlantic salmon is motivated by procreation and recruitment of successive generations. With this in mind, it is not surprising that reproductive investment occurs very early in the Atlantic salmon life cycle, with differentiation of germinal tissue occurring prior to first feeding during the embryo stage of development (Mangel and Satterthwaite, 2008). In this context, some scientists have described the general process of maturation as being controlled by inhibition during the juvenile life stages until a specific physiological threshold is reached that triggers a developmental switch (Thorpe, 1986; Thorpe et al., 1998; Mangel and Satterwaite, 2008). Proposed thresholds include: level of adipose tissue (Rowe and Thorpe, 1991; Simpson, 1992), size or weight of the fish (Skilbrei, 1989; Shearer et al., chinook salmon, Oncorhynchus tshawytscha), condition factor (Herbinger and Friars, 1991; Peterson and Harmon, 2005), and energy/nutrient reserves (Kadri et al., Page 11

12 1996), all of which provide information about optimal fitness, and the likelihood of successful survival and reproduction following a rigorous migration back to natal spawning waters (Mangel and Satterwaite, 2008). A number of literature sources indicate that the maturation trigger appears to depend on physiological or biochemical conditions (the aforementioned thresholds) that are to some degree genetically determined but also influenced by environmental factors (Naevdal, 1983; Gjerde, 1984; Saunders, 1986; Thorpe et al., 1998; Mangel and Satterwaite, 2008; Taranger, 2010). Saunders (1986) proposed that the genetic influence on maturation provides a basis for maturation but with rather wide latitude, not necessarily preset for a specific time or age but instead expressed when the appropriate environmental and physiological/biochemical conditions are met. A similar synopsis was provided by Mangel and Satterwaite (2008) who described optimization of environmental conditions, such as water temperature, as creating an opportunity for growth along with other traits that typically parallel optimal growth performance, such as the accumulation of adipose tissue. Although the exact mechanisms of the onset of maturation are not fully understood in Atlantic salmon, this literature review indicates that the process of maturation is likely triggered somewhere within the boundaries of a variety of heritable, physiological/biochemical, and environmental cues, and their interactions. This introductory review also emphasizes the high degree of plasticity of life history traits among and within Atlantic salmon populations, and demonstrates that the life cycle of the Salmo salar is highly motivated by reproduction. When considering the complexities of Atlantic salmon life history, it becomes clear that controlling maturation within aquaculture systems is a complex task. Possible solutions to early maturation, however, Page 12

13 likely begin within an understanding of the Atlantic salmon s inclination for reproductive advantage and subsequent identification and control of the environmental and physiological cues that trigger reproductive development. FACTORS INFLUENCING MATURATION Photoperiod Photoperiod is considered an essential determinant for initiating sexual maturation in teleosts (Taranger et al., 2010). Its singular importance, which is rooted in the evolution of reproductive strategies of upper latitude fish species, is to ensure hatching of juveniles during periods of advantageous environmental conditions (Bromage et al., 2001). The effect of photoperiod on salmonid maturation has been extensively studied, and its interaction with water temperature has become an area of increased focus (i.e. Fjelidal et al., 2011; Imsland et al., 2014). Based on decades of research on salmonids and other species, annual physiological rhythms are thought to be entrained with seasonal changes that are sensed through periods of increasing or decreasing daylength, and sexual maturation is initiated or postponed during a critical time window based on numerous other factors, such as size, growth rate, nutritional status, and genetics (Duston and Saunders, 1992; Taranger et al., 1998; Taranger et al., 1999; Bromage et al., 2001; Taylor et al., 2008; Taranger et al., 2010). The direction of photoperiod change, as opposed to the specific day-length, is considered most important in orchestrating sexual maturation and the eventual seasonal timing of reproduction (Bromage and Duston, 1986; Bromage et al., 2001). In manipulating photoperiod to reduce the proportion of fish switched on to undergo puberty, artificial short days have been used during the early months of the year when natural photoperiod is otherwise increasing, followed by artificial long days after midsummer when natural Page 13

14 photoperiod declines (Randall et al., 1998; Bromage et al., 2001); however, prolonged exposure to long days, i.e. into the final month of the calendar year, could potentially increase the percentage of fish that sexually mature (Duncan et al., 1999). Towards harvest, however, continuous light is routinely applied in the net pen industry from winter to summer solstice to prevent maturation in salmon during their second year at sea (Leclercq et al., 2011). Closed containment aquaculture allows for greater control of environmental conditions compared to open systems, and as such, photoperiod regimes can be easily applied throughout the production cycle, from hatch to harvest, while keeping other environmental variables (e.g. water temperature) relatively constant. The majority of research on developing photoperiod regimes to prevent early maturation, however, has been carried out in the context of early rearing in land-based, freshwater systems, followed by the transfer of smolts to sea cages. While photoperiods can be manipulated in both preand post-transfer rearing environments of the traditional culture scheme, other important variables (e.g. salinity) can obviously be widely different throughout the production cycle compared to land-based closed containment systems. Research strictly focused on photoperiod manipulation in landbased growout environments has been extremely limited, and much more work is needed in this regard to better understand fish physiology in closed containment systems in order to develop best management practices for reducing or eliminating early maturation. Consideration must also be given to photoperiod manipulation in the context of bioprogramming, given the potential simultaneous presence of multiple age classes and continuous production due to year-round eyed egg availability, which could present a new set of challenges beyond those faced in operations using a traditional salmon production cycle. Page 14

15 For purposes of this paper, photoperiod will be abbreviated in the form of LDX:Y, where LD stands for light-dark and X:Y is the amount of light (X) and dark (Y) hours over the course of a 24-hour period; for example, LD16:8 refers to a photoperiod in which fish are exposed to 16 hours of light followed by 8 hours of darkness. Good et al. (2015) demonstrated that Atlantic salmon exposed to a reduced photoperiod, i.e. LD18:6, from first feeding up to oneyear post-hatch in freshwater RAS, exhibited a significantly higher proportion of mature males than those exposed to continuous light during their first year. The impetus for examining a reduced photoperiod, versus continuous light, and its effects on early maturation, was twofold: (i) Atlantic salmon cohorts raised to market size at TCFFI under continuous light (excepting a six-week LD12:12 winter to synchronize smoltification, beginning at 40 g average weight), have consistently produced a high percentage of maturing males, whereas the most affected group demonstrated approximately 80% male maturity before the mean population weight reached 2 kg; and (ii) previously published research suggests that a reduced photoperiod can reduce early maturation compared to continuous light. The latter research includes the study by Fjelldal et al. (2011), in which significantly higher proportions of mature males were observed in populations exposed to three months of early rearing continuous light, versus those exposed to a natural photoperiod; however, further analyses suggested that the continuous light treatment was acting as an enabling factor, while elevated water temperature determined the actual degree of early maturation observed. The study by Good et al. (2015) found the opposite effect of reduced photoperiod during early rearing (but at 13 C during this treatment period, versus 16 C in the Fjelldal et al. (2011) study, and in fresh water as opposed to seawater). The findings by Good et al. (2015) resemble those of Berg et al. (1996), who found higher maturation in salmon exposed Page 15

16 to LD20:4 photoperiod from smolt to harvest size, versus those exposed to LD24:0, in marine net pen environments. Given the differences in treatments, photoperiod exposure times, and environment conditions, it is difficult to directly compare these (and other) experiments. The study by Good et al. (2015), however, was carried out under conditions similar to those provided in current closed containment salmon growout operations, and therefore the study results, although limited, can be considered of higher relevance to closed containment Atlantic salmon production, and can serve as a basis for further research. Because the decision to mature is likely made during the first year posthatch, when Atlantic salmon smolts in the traditional industry are often in freshwater recirculation systems, previous research within this context is relevant to closed containment producers. For example, Saunders and Henderson (1988) compared four photoperiod treatment groups, LD24:0, LD16:8, LD12:12, and LDN (simulated natural photoperiod), with fish exposed from first feeding (May) until the following January, to determine differences in precocious parr prevalence. Although precocious parr were prevalent in all groups (ranging from 43.9% to 66.7%), LDN fish had a significantly higher proportion of mature parr, while the LD16:8 had significantly less mature parr than the other treatment groups. While precocious parr have not tended to be a significant issue during the early stages of TCFFI growout trials, mature underyearling smolts were observed in very high numbers from a portion of one growout population that was transferred to six replicated experimental RAS for a parallel study. Whether this unusually high incidence of early maturation was due to sudden exposure to warmer water temperatures (approximately 2 C increase), unintentional photoperiod change, or Page 16

17 other environmental cues, remains unknown. The results of Saunders and Henderson s (1988) study are interesting in that the authors found a significant decrease in mature salmon exposed to a reduced photoperiod during the first year, compared to a constant photoperiod, which is opposite to the relationship determined by Good et al. (2015); it is unfortunate that growth in freshwater up to market size was not a consideration for the study by Saunders and Henderson (1988), as a full comparison between studies cannot be carried out. Thorpe (1986) proposed a unified model to study the interactions of salmon growth, smolting, and maturation rates based on observations of precocious parr in the Scottish salmon industry. This model was tested by Adams and Thorpe (1989) by comparing underyearling fish exposed to 2x2 factorial treatments of either elevated or ambient water temperatures, and either advanced (3-months ahead of natural phase) or natural, ambient photoperiods. As predicted by Thorpe (1986), conditions favoring growth (i.e. increased water temperature) during a February maturation window were associated with increased parr maturation. Enhanced parr maturation was avoided in other treatment groups, particularly for fish exposed to elevated water temperatures with an advanced photoperiod, such that they did not experience the maturation window of early-year ambient day-length increases. These findings emphasize the importance of photoperiod control during very early salmon development, in particular avoiding parr exposure to ambient photoperiod. Avoiding ambient photoperiod is relatively easy to achieve in closed containment aquaculture operations that use enclosed buildings; however, if exposure to ambient light during the maturation window cannot be guaranteed (e.g. when procuring young salmon from outside facilities using Page 17

18 assumed or unknown photoperiod conditions), other methods can be used to suppress early maturation. For example, parr maturation has been shown to be switched off by growth suppression during the spring months, such as by fasting fish in alternate weeks during this period (Rowe and Thorpe, 1990). Beyond the parr stage, Atlantic salmon smolts have the capacity to sexually mature early as grilse, which can be a major challenge to the traditional industry as these fish (unlike precocious parr) cannot be adequately identified and culled out prior to sea cage transfer. A variety of photoperiod treatments have been applied to sea cages, using underwater lighting, to reduce or eliminate grilsing. It must be noted that supplemental lighting in sea cages cannot be compared directly to lighting in closed containment growout conditions, as salmon in sea cages can still sense changes in ambient photoperiod beyond the artificial light being applied. Thus, continuous photoperiods employed in sea cages should be considered as continuous additional lighting, versus a true LD24:0 photoperiod that can be applied in closed containment conditions. An interesting study by Taranger et al. (1998) compared the effects of nine different photoperiod regimes applied to immature Atlantic salmon in their final year of production (i.e. following 1.5 years at sea under natural photoperiod conditions, and a grilse cull prior to study initiation) on the incidence of early maturation. In this study, fish were raised in sea cages until mid-summer, and then transferred to landbased raceways using brackish water. Photoperiod treatment combinations included exposure to (i) natural light, (ii) continuous additional light beginning in January, or (iii) continuous additional light beginning in March, while in sea cages, and (i) natural light, (ii) LD24:0, or (iii) LD8:16 following transfer to raceways. All treatment groups receiving either natural light or continuous Page 18

19 additional light beginning in March while in sea cages exhibited high (>50%) grilsing rates, with the highest grilsing rate (78%) observed in the treatment group receiving natural photoperiods in both sea cages and raceways. By far, the lowest grilsing (5%) was observed in fish receiving continuous additional lighting beginning in January while at sea, and LD24:0 while in raceways. Again, while it is difficult to fully extrapolate these results to closed containment growout conditions, these findings suggest that continuous photoperiod during the final year of production can assist in decreasing overall grilsing rates. However, because LD24:0 photoperiods have been applied during TCFFI salmon growout trials, with correspondingly high, but variable grilsing rates, there are clearly other factors that could be influencing maturation in these trials, such as water temperature in the growout phase, photoperiod conditions during first-year rearing prior to growout, or other unknown risk factors. Research on photoperiod manipulation immediately following the induction of smoltification, i.e. an artificial winter followed by a period of LD24:0, has been carried out by Duncan et al. (1999), who compared maturation rates in post-smolts transferred to seawater (in this case, land-based tanks with pumped-ashore ocean water) and monitored for a year under various photoperiod conditions. Surprisingly, first-year post-smolts receiving LD24:0 for one year starting in December, (whereas, photoperiod conditions did not change following the induction of smoltification), demonstrated the highest maturation rates, while salmon exposed to a simulated natural photoperiod demonstrated no maturation, based on a GSI cut-off of 3%. Fish in the LD24:0 group exhibited significantly greater growth during the study year, although specific growth rate (%/day) declined in comparison to the simulated natural Page 19

20 photoperiod group during the final months of the year. Given that the natural photoperiod during this timeframe represented a true calendar photoperiod (i.e., spring increase, followed by fall decrease), it would have been informative to have included a treatment group receiving natural photoperiod until summer solstice, followed by LD24:0 for the remainder of the year; this change to LD24:0 following midsummer could have switched off the development of puberty in the second half of the calendar year (Bromage et al., 2001). Nonetheless, the results of this study could have implications for closed containment production, in that a period of increasing daylength following induction of smoltification might decrease the incidence of early maturation observed during the remaining production period. Berrill et al. (2003) investigated the timing of applying an artificial winter to induce smoltification on the subsequent incidence of precocious parr maturation, considering that faster growing fish would decide whether to devote energy to smoltification or sexual maturation. Fish were given LD24:0 photoperiod from first feeding onwards, and an S 0 winter either beginning in May, August, or September (or, in a fourth group, no S 0 winter was applied), followed by a return to LD24:0 photoperiod. Fish exposed to an early (May) winter had significantly higher precocity versus other groups, whereas late artificial winter groups (August and September) had relatively low maturity levels (although fish in the September group did not smoltify as completely as those in the August group). The S 0 winter is typically applied at TCFFI beginning in August; however, direct comparison between TCFFI maturation rates and those observed by Berrill et al. (2003) is difficult, due to (i) fish size differences (generally 40 g in average weight prior to S 0 winter at TCFFI, vs <10 g as reported by Berrill et al. (2003)), and (ii) water temperature Page 20

21 differences, whereas TCFFI fish are typically exposed to C water during first year rearing, versus Berrill et al. (2003) who exposed fish to ambient temperatures ranging from approximately 20 C in midsummer to <5 C in January. Therefore, in this study, fish exposed to different S0 winters were also exposed to different water temperatures, and different directions of water temperature change, during these winters. In a follow-up study under similar conditions, Berrill et al. (2006) determined that a longer (i.e. 12-week, versus 8-week) short-day period to induce smoltification, starting in June as opposed to May, significantly reduced the number of precocious parr observed later in the year. Again, while the results of these studies are ultimately difficult to compare to observations made during TCFFI growout trials, they clearly demonstrate the importance of early rearing environmental factors, i.e. photoperiod, temperature, or the interaction of these two parameters, on subsequent precocious maturation. Thus, emphasis in future research should be applied to first-year environmental conditions as maturation decisions are clearly made beginning at a very early age. While much research has focused on the effects of various photoperiod regimes throughout the Atlantic salmon production cycle, a comparatively small volume of research has examined the quantity (light intensity) and, in particular, the quality (spectral composition) of artificial light that fish are exposed to within these photoperiod regimes. Both quantity and quality of light have been shown to affect growth, reproduction, and other performance variables in teleosts (Oppedal et al., 1997; Karakatsouli et al., 2007, 2008). Light intensity, in particular, appears to act in a threshold manner in regulating various physiological functions in fish (Porter et al., 1999; Taylor et al., 2005, 2006), and increasing light intensity beyond a specific threshold has been Page 21

22 shown to increase growth and decrease maturation in typical end-of-cycle constant-light photoperiods applied to Atlantic salmon (Stefansson et al., 1993; Oppedal et al., 1997, 1999); however, decreased welfare associated with high intensity lighting has been noted (Migaud et al., 2007; Vera and Migaud, 2009). Based on studies using the hormone melatonin as an indicator for light perception (i.e. with increased light levels, melatonin release by the pineal gland is reduced), the light intensity threshold for perception in Atlantic salmon appears to be around W/m 2 (Migaud et al., 2006; Vera et al., 2010). In terms of light quality, studies have suggested that Atlantic salmon suppress melatonin production more efficiently in response to blue and green light (450nm and 550nm, respectively) compared to red light exposure (700nm) (Migaud et al., 2010; Vera et al., 2010); however, more research is needed on the spectral sensitivity of Atlantic salmon. In a recent study, Leclercq et al. (2011) examined different lighting strategies to determine, among other things, their respective efficacy at controlling sexual maturation in Atlantic salmon during sea cage growout. While the effects of spectral composition (blue, red, green, or broad spectrum) could not be distinguished from light intensity, the authors data strongly suggest that light intensity is the major determinant affecting Atlantic salmon light perception (and hence, affecting suppression of sexual maturation), and that a mean intensity of W/m2 appeared to be the threshold to reduce maturation (which coincides closely with the W/m2 threshold intensity determined in laboratory studies, mentioned earlier). Given the differences in closed containment versus sea cage growout conditions (e.g. rearing unit volume, water clarity, rearing densities, etc.), baseline research is needed to establish lighting quantity and quality best management practices in order to reduce early maturation while not compromising welfare in Atlantic salmon closed containment production facilities. Page 22

23 Input from industry TCFFI has, for the most part, employed a LD24:0 photoperiod from first feeding up to harvest at 4-6 kg in size using above-tank metal halide, fluorescent, and LED lights, excepting a pre-smolt LD12:12 6-week winter, and has experienced varying but relatively high levels of grilsing in all Atlantic salmon growout cohorts. Several Atlantic salmon producers were contacted for the purposes of this technical paper, regarding their current photoperiod strategies and relative success at reducing grilsing. The following is a summary of anecdotal information generously provided by these individuals. Danish Salmon (Hirtshals, Denmark) employs the same photoperiod strategy as TCFFI (i.e. LD24:0 throughout, aside from a LD12:12 winter to induce smoltification), although due to numerous water quality issues (as a consequence of chilling and discharge consents) over the past year, they are reluctant to make any conclusions regarding the impact of their photoperiod regime on maturation (Mark Russell, Danish Salmon, pers. comm.). Sustainable Blue (Centre Burlington, Nova Scotia, Canada) has carried out in-house research to reduce maturation in their salmon, utilizing a LD16:8 photoperiod and focusing on feeding rates within this regime; Jeremy Lee provided the following description of this work: We performed an initial trial on our first batch of salmon [smolts from an off-site producer, g in size]. These were St John River strain. The batch was split into two tanks on arrival from a local hatchery; roughly 50% per tank. One tank was held on 75% ration and the second on 100% ration for the first 100 days in saltwater. After 100 days both tanks were fed 100% planned Page 23

24 ration. Both tanks were at 15 C and on the same water treatment system. Both tanks had a 16 hour daylight, 8 hour dark photoperiod. Total maturation was 8% at 9.5 months and an average weight of 2.8 kg. Importantly there was no significant difference in maturation between the tanks at the 95% confidence level. It is still too early to report on our latest batches. These are from our own hatchery and have been on controlled photoperiods throughout their lives. Those in the grow-out unit are currently on a 16 hour day, 8 hour night; the same that was used for the initial batch. The largest batch has now reached 1 kg after 3.5 months in 26 ppt. There is no sign of maturity so far and the water temperature is 14 C. This stock is Mowi. (Jeremy Lee, pers. comm.) Frode Mathisen, Director of Biological Performance and Planning for Grieg Seafood ASA (Bergen, Norway), provided the following information on photoperiods applied either through their freshwater or seawater regimes, prior to transferring smolts to sea cages: We haven t seen any maturation issues [no maturation in hatcheries and very low grilsing in sea cages]. We run two different light regimes, depending on the salinity the hatchery can run: Freshwater regime gram (±20 g) on 24 hour light gram (±20 g) up to 3-4 weeks before delivery to sea: on 12 hour light (minimum time on 12 hour light is 4 weeks) 3. The last 3-4 weeks on 24 hour light until the fish finish smoltification and are delivered to seawater sites In this regime it is important to turn the lights off before the fish go into a size smoltification. There is some uncertainty about that process, but Page 24

25 we have kept fish up to 70 gram on 24 hour light without any significant smoltification issues. When the fish are on 12 hour light, we can hold it there as long as we want. With this process we have produced fish groups from 60 to 250 gram. Seawater regime gram (±20 g) on 24 hour light gram (±20 g): 4-6 weeks on 12 hour light 3. Fish are moved into brackish RAS on 24 hour light (salinity 8-15 ) where the fish finish smoltification in 3-4 weeks. Some fish are moved to seawater sites as soon as they have become smolts Most fish are kept in the brackish RAS on 24 light until they reach gram and are delivered to a seawater site Lastly, through a 16-month production cycle Kuterra Land-Based Salmon (Port McNeill, BC, Canada) has generally employed a LD24:0 photoperiod from winter to summer solstice, and simulated natural photoperiod from summer to winter solstice, although this strategy has varied depending on the time of year that various smolt cohorts have arrived on-site. Grilsing (both male and female) has generally been high, and the percentage of grilse has varied among growout cohorts (Cathal Dineen, Kuterra, pers. comm.). Water temperature Water temperature is one of, if not the most important environmental parameter known to influence fish physiology. Fish are poikilothermic, meaning that their internal temperature is largely regulated by the ambient temperature of the environment; thus, water temperature has a profound influence on Page 25

26 fish biology. Metabolic rate, growth rate, time of spawning and egg hatching, the physiological development of eggs and larvae, and the reproductive development of fish are all related to water temperature of the natural environment (Piper et al., 1982; Jobling, 1995; Wedemeyer, 1996). The effect of water temperature on Atlantic salmon biology is no exception. Brett (1979) reported that salmonid growth increases linearly with temperature. Research provided by Austreng et al. (1987) supports these findings, demonstrating that young Atlantic salmon cultured in freshwater from g at temperatures ranging from 2-16 C, grew fastest at 16 C and slowest at 2 C. The same study reported maximum Atlantic salmon growth in marine net cages at 14 C, when evaluating fish growing from kg at temperatures ranging from 2-14 C (Austreng et al., 1987). Handleland et al. (2008) reported that growth rate, feed intake, feed conversion efficiency, and stomach evacuation rate were significantly influenced by water temperature (6-18 C) and size of post-smolt Atlantic salmon ( g) cultured in seawater, with the fastest growth rates occurring at 14 C and the slowest growth rates occurring at 6 C. The importance of water temperature to Atlantic salmon biology is amplified when considering the seasonal migratory behavior of this species. Jonsson and Rudd-Hansen (1985) found that temperature acted as the primary parameter of influence for downstream smolt migration from the Imsa River in Norway, while McCormick et al. (2002) concluded that water temperature likely controlled the rate of juvenile development, but interacted with photoperiod relative to the timing of smoltification. Friedland et al. (2000) reported that Atlantic salmon smolts from the Figgio River in Norway and North Esk River in Scotland typically swim to sea during late April to early May when the marine water temperature is 8-10 C. Furthermore, a wealth of studies (described in the subsequent section) have linked temperature to Atlantic salmon size/ age at maturation, the timing of reproductive maturity, and the proportions of grilse versus multiple sea-winter salmon. Page 26

27 Saunders et al. (1983) cited substantial evidence of the water temperature of sea-cage sites acting as a determinant of the timing of Atlantic salmon maturation, where lower water temperatures often correlated with reduced maturation during the first sea-winter and a decreased rate of grilsing. In addition, Adams and Thorpe (1989) found that female Atlantic salmon exposed to increased water temperature, 5 C above typical ambient temperature, showed higher reproductive investment (ooycyte size) than those under normal growing conditions, and also found that male parr exposed to temperature conditions consistent with an increased growth opportunity had a higher maturation rate. Several recent studies provide additional evidence that increased water temperature is at least partly related to early maturation of Atlantic salmon. During a long-term study comparing different combinations of light and water temperature, Imsland et al. (2014) observed an increased rate of early maturing male Atlantic salmon (i.e. 66% vs. 11%) when cultured at 12.7 C vs. 8.3 C, respectively. During this study, pre-smolt Atlantic salmon (initial weight = 15.9 g) were cultured in freshwater for 11 months, then relocated to seawater for 2 months. After the 2-month seawater period, the salmon were slaughtered to assess final weight ( g) and maturity. Atlantic salmon cultured under continuous light at higher water temperature (12.7 C) grew % faster than other groups, but also exhibited the highest degree of early male maturation (82%). Imsland et al. (2014) concluded that long term rearing of Atlantic salmon under continuous light, but lower water temperature (in this case 8.3 C) led to a better balance of growth and reduced maturation. Based on these findings, Imsland et al. (2014) suggested that photoperiod was the primary directive for the onset of sexual maturation, but temperature likely controlled the magnitude of the photoperiod effect. Similarly, Fjelldal et al. (2011) demonstrated that a combination of elevated water temperature and Page 27

28 continuous light can trigger maturation of male Atlantic salmon during and immediately after smoltification. Early maturation of male Atlantic salmon was particularly pronounced for parr cultured at 16 C with a 24-h photoperiod, as compared to culture at 5 and 10 C in combination with various photoperiod regimes, whereas 47% of male salmon cultured at 16 C began to mature (i.e. mean gonadosomatic index (GSI) > 1.5%) after only 6 weeks of exposure. After an additional 2 months of culture, the same population of maturing males was found to have a mean GSI of 7.3%. No maturing males were noted after the initial 6-wk trial for temperature treatments of 5 and 10 C combined with any of the photoperiods (including 24-h continuous light) (Fjelldal et al., 2011). With so many documented variables and combinations of parameters that could influence maturity, separating the most impactful factors is a complex task. Therefore, McClure et al. (2007) applied a multivariate approach to identify variables that were most associated with greater risk of grilsing in Atlantic salmon. Grilsing prevalence was evaluated at 266 commercial net cage sites in New Brunswick and Nova Scotia, at 24 different farms. The percentage of grilse within cages was variable, ranging from %; while the withinfarm rate of grilsing ranged from %. The median within-cage and -farm grilsing rate was 6.6% (McClure et al., 2007). A variety of risk factors possibly contributing to increased grilsing were evaluated during the McClure et al. (2007) study including: smolt weight at time of transfer to net cage, cage type, use of moist feed and duration of feeding moist pellets, feeding rate, weight gain, average water temperature during the first February at sea, average water temperature during second September at sea, and the change in water temperature between the first February and second September at sea. The final statistical model identified two risk factors that were most associated Page 28